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Background: Nitric oxide (NO), a vasodilator molecule, is produced from L-arginine by endothelial nitric oxide synthase (eNOS) and it regulates endothelial function. The presence of eNOS variants might further complicate endothelial dysfunction and nephropathy through reduced production of NO. This study aimed to find out the possible association and synergistic effect of eNOS 4a/b and G894T polymorphisms on the risk of diabetic nephropathy. Patients and Methods: This study conducted on 45 patients with diabetes mellitus type 2 and 15 healthy subjects serving as a control group. All candidates were genotyped for the eNOS polymorphism using PCR-RFLP technique. Results: there are significant differences in genotype and allele frequencies between patients and control for eNOS G894T and (4a/b) Polymorphisms with p value ˂0.05, and It also showed significant differences between patients with DN and patients without DN for the GT genotype, a and T alleles with p value ˂0.05. The subjects with GT or aa genotypes were associated with risk to develop micro and macroalbuminuria when compared with controls but the presence of both them had no significant effect. Conclusion: Analyzing both eNOS 4a and 894T alleles revealed that in the presence of either eNOS 4a or 894T allele, the risk of developing DN increased significantly. However, the presence of both alleles together was not associated with increased risk of microalbuminuria and macroalbuminuria.